Faculty Bibliography

UNLABELLED:mutant did not develop abscesses past early stages. These results have implications for the genes regulated by Agr and Sae and suggest a requirement for Sae activity during kidney abscess development. IMPORTANCE/OBJECTIVE:, Agr and Sae. While Agr appeared inactive during kidney abscess development, the Sae system exhibited heterogeneity, increased expression at later stages, and was required for abscess progression. These results provide critical information for the development of virulence factor-targeting strategies for kidney abscess treatment.

Rapid molecular assays guiding treatment of methicillin-resistant Staphylococcus aureus (MRSA) detect SCCmec (Xpert) or the SCCmec-orfX junction (BCID2). Sequence variation in this region can disrupt primer binding, yielding false-negative results. Investigation of a missed bloodstream infection linked escape to a CRISPR-Cas-associated SCCmec variant, leading to identification of 64 variants from 45 patients-2% of 2,432 screened. Misdiagnosis was restricted to clonal complex 5, a hospital-associated lineage; 11 of 40 SCCmec/junctions evaded detection by BCID2 or Xpert. Variants had mecA instability and circulated in healthcare settings. Our findings reveal a unique escape mechanism and underscore a threat to diagnostic accuracy.

Asymptomatic carriers of antimicrobial-resistant organisms (AMROs) can unwittingly transmit these pathogens in hospitals, contributing to the burden of healthcare-associated infections (HAIs). Surveillance in hospitals can involve different types of observations; however, a framework to coherently synthesize these datasets to identify AMRO carriers is lacking. Here, we develop a new inference framework combining a data-driven mechanistic transmission model and multimodal observations from clinical cultures, electronic health records, patient mobility, and genomic data. Using extensive simulated outbreaks, we validate the inference framework for AMROs with various levels of community importation and hospital transmission and evaluate the utility of different combinations of data sources. Inference results show that using multimodal observations consistently improves the accuracy in identifying AMRO carriers. We apply the inference framework to carbapenem-resistant Klebsiella pneumoniae (CRKP) at an urban quaternary care hospital in New York City, United States and find that the addition of even sparsely sampled genome sequence data to patient characteristics supports more accurate identification of CRKP carriers. Model simulations suggest that inference-guided targeted isolation leads to a greater reduction of AMRO burdens compared to alternative, heuristic approaches. Thus, the synergistic effect of utilizing multimodal observations for estimating AMRO carriage risk may inform improved interventions in hospital settings.

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present report shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding a known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a critical mechanism-epigenetic regulation of staphylococcal gene expression-by which phage can regulate virulence to drive adaptive leaps by S. aureus.

Staphylococcus aureus is a global health concern, resulting in significant disease burden in both hospital and community settings. To establish infection, the bacteria must contend with a multitude of host defense mechanisms, including "nutritional immunity", in which nutrients are sequestered away from invading pathogens. Importantly, S. aureus requires iron for growth during infection, which it acquires through the lysis of erythrocytes (hemolysis). HlgAB, a secreted bi-component pore forming toxin, contributes to the ability of S. aureus to lyse erythrocytes to release heme iron. HlgAB consists of two subunits, the S-subunit HlgA and the F-subunit HlgB. Prior work has shown that the hemolytic activity of HlgAB is dependent on the binding of HlgA to the host receptor Duffy Antigen Receptor for Chemokines (DARC). Here we show that HlgB binds the surface of erythrocytes independently of DARC or HlgA. Our comparative genomic analysis reveals high conservation of hlgA and hlgB genes across S. aureus lineages. By performing structure-function studies, we identified a series of loops within the rim domain of HlgB that are required for the binding of HlgB to erythrocytes and erythrocyte lysis by HlgAB. The importance of HlgB-mediated host targeting was validated in a tissue culture model of S. aureus-mediated lysis of primary human erythrocytes, in an in vivo murine model of intoxication, and during in vivo systemic infection. Altogether, these findings expand our mechanistic insights into how S. aureus overcomes nutritional immunity, and the role of HlgB in S. aureus pathophysiology.

Pathogens have evolved to be highly adapted to their natural host. Community-associated methicillin-resistant Staphylococcus aureus USA300, for instance, is a lineage responsible for the epidemic of skin and soft tissue infections (SSTIs) in humans. Owing to its human tropism, mechanisms that enabled the rise of USA300 as a major skin pathogen remain incompletely defined. By leveraging a rodent-adapted strain of S. aureus, we developed a natural model of SSTIs. We found that LukMF', a pore-forming leukocidin homolog to the human-specific LukSF-PV toxin, drives skin pathology in mice. LukMF' lyses neutrophils via the chemokine receptor CCR1, which in turn fuels inflammatory pathology and microbial survival within the infectious nidus. Ablation of CCR1, depletion of neutrophils, or vaccination with LukMF' all protected mice from skin pathology. Thus, these data support epidemiological studies linking leukocidins with human SSTIs and highlight the power of natural models to unearth potential targets to curtail infections.

In the human pathogen Staphylococcus aureus, the two-component regulatory system SaeRS contributes to the expression of numerous virulence factors essential for pathogenesis. The kinase and phosphatase activities of SaeS are stimulated by several host and physiological signals, resulting in increased phosphorylation of the transcription factor SaeR and increased transcriptional activity of regulated promoters. It was recently demonstrated that the accumulation of fatty acids negatively impacts SaeS activity, decreasing titers of phosphorylated SaeP and transcriptional output. Triclosan is an effective antimicrobial that has been integrated as an ingredient in a variety of healthcare and consumer products. The chlorinated compound is recalcitrant to natural or biological transformations, resulting in environmental accumulation. At low concentrations, triclosan is a bacteriostatic inhibitor of enoyl-acetyl carrier protein reductase (FabI) of the type II fatty acid synthesis system (FASII), which is necessary for the elongation and synthesis of fatty acids. Herein, we demonstrate that the treatment of S. aureus with a growth-permissive concentration of triclosan alters the titers of cell-associated fatty acids and thereby functions as an activator of SaeRS. Triclosan-dependent activation of SaeRS subsequently resulted in increased transcription and expression of genes that code for virulence factors. These phenotypes are chemically reversed by the exogenous addition of oleic acid, which inactivates SaeRS, and genetically reversed by crippling the FakAB fatty acid kinase system, which generates phosphorylated fatty acids for incorporation into phospholipids. These findings present implications for the widespread use of triclosan as an antimicrobial agent in household products and its role as a persistent environmental pollutant.

The T300A substitution in ATG16L1 associated with Crohn's disease impairs autophagy, yet up to 50% of humans are heterozygous for this allele. Here, we demonstrate that heterozygosity for the analogous substitution in mice (Atg16L1^T316A), but not homozygosity, protects against lethal Salmonella enterica Typhimurium infection. One copy of Atg16L1^T316A was sufficient to enhance cytokine production through inflammasome activation, which was necessary for protection. In contrast, two copies of Atg16L1^T316A inhibited the autophagy-related process of LC3-associated phagocytosis (LAP) and increased susceptibility. Macrophages from human donors heterozygous for ATG16L1^T300A displayed elevated inflammasome activation while homozygosity impaired LAP, similar to mice. These results clarify how the T300A substitution impacts ATG16L1 function and suggest it can be beneficial to heterozygous carriers, providing an explanation for its prevalence within the human population.

Staphylococcus aureus is a major cause of bacterial infection-related deaths. Increasing antimicrobial resistance highlights the urgent need for effective preventative strategies. Antibody-mediated opsonophagocytosis, the key mechanism for protection against S. aureus, is disabled by critical virulence factors such as Staphylococcal protein A (SpA) and leukocidin AB (LukAB). In our study, we combined genetically detoxified vaccine candidates SpA* and LukAB RARPR-33 with a T_H1 adjuvant aiming to restore host antibody functionality. To evaluate these vaccine candidates, we developed both surgical site infection (SSI) and superficial wound infection (SWI) models in minipigs. Our results showed a significant reduction in bacterial load and systemic dissemination in the SSI model, while skin infection severity was markedly decreased after intradermal immunization in the SWI model. This study introduces a novel S. aureus vaccine strategy by targeting immune evasion factors SpA and LukAB, utilizing potent T_H1 adjuvants, and employing minipig challenge models.